1. Field of the Invention
The present invention relates generally to polymer bound compounds such as 1-hydroxybenzotriazole-6-carboxylic acid (P-HOBT) and uses thereof. In particular, the present invention relates to a device for the single-step amino group modification of proteins, peptides, and amines via acylation reactions. The present invention also relates to a method of detecting the presence of amines in a sample using polymer bound compounds such as 1-hydroxybenzotriazole-6-carboxylic acid.
2. Description of the Related Art
Combinatorial chemistry has become one of the major tools in the search for new lead compounds in pharmaceutical drug discovery (1-3). As the use of combinatorial chemistry increases, much effort has gone into the development of new synthetic methods for the highly efficient preparation of chemical libraries. One of the results of this research has been an increase in the development and use of solid-phase organic synthesis techniques.
Two general protocols have been used in solid-phase organic synthesis: (i) the use of polymers as a scaffold upon which to build the desired molecule in a multi-step sequence, or (ii) the use of a polymer-supported reagent to mediate a single synthetic step. The use of polymers as templates for building complex molecules has been exploited in well-known applications such as solid-phase peptide synthesis (4) and oligonucleotide synthesis (5).
Beyond these technologies, a variety of organic reactions have been successfully carried out in the solid phase (3, 5-11). Moreover, this methodology has been used for the synthesis of small heterocyclic organic molecules, including benzodiazepines, diketopiperazines and hydantoins (12-14).
Alternately, the use of polymer supported acylating agents as catalysts has been particularly useful for the formation of amide bonds, as well as ester formation. These immobilized reagents include nitrophenol (15-17), N-hydroxysuccinimide (NHS) (18), 1-hydroxybenzotriazole (HOBT) (19-21), carbodiimide (22, 23), and triphenylphosphine (24, 25). Of these polymer supported acylating agents, polymer supported HOBT (P-HOBT) has been the most useful. P-HOBT has been used as a highly reactive N-acylating agent for the formation of peptide bonds (19-21) and simple amides (26, 27). P-HOBT has also been utilized for the synthesis of medium-ring lactams from linear ω-amino acid precursors (28), as an acylating agent for the synthesis of NHS esters (29), and for the carbamate protection of primary and secondary amines (30). Excluding solid-phase peptide synthesis and organic synthesis, current protein, peptide, and amine modifications are carried out in solution phase reactions which involves subsequent work-up and/or purification of the product after the reaction.
In the work of Fridkin and Patrchomik (19), the immobilized HOBT reagent was synthesized by the direct functionalization of polystyrene utilizing 4-chloro-3-nitrobenzyl alcohol under Friedel-Crafts conditions. The HOBT moiety was formed by treatment of the polymer with hydrazine monohydrate and subsequent cyclization under acidic conditions using known methods (31). Although this method has been utilized for the preparation of P-HOBT, it is limited to polystyrene (aryl) based polymers, and is not always reproducible due to the Friedel-Crafts reaction, the synthesis of P-HOBT was improved by the initial coupling of 4-chloro-3-nitrobenzenesulfonyl chloride to an aminomethylated polystyrene polymer, followed by reaction with hydrazine monohydrate and cyclization under acidic conditions (26). The polymer is highly active due to the presence of the electron withdrawing sulfonyl group in the benzene ring, and can by synthesized in a reproducible manner. A disadvantage of this approach is the two step method of HOBT cyclization after addition of the sulfonyl chloride to the polymer, utilizing conditions which may not be compatible for the derivatization of all solid supports. This limits the choice of solid supports used to immobilize the HOBT and requires the adaptation of an application to utilize a polymer support that can be modified using the heretofore known method of coupling 4-chloro-3-nitrobenzoic acid to the polymeric support, followed by the two step formation of HOBT.
Thus, before the present invention, there were no known effective means of directly immobilizing 1-hydroxybenzotriazole or a derivative thereof to a solid support under mild conditions, i.e., in organic or aqueous solvents at neutral pH and at room temperature. Additionally, there were no known methods of using immobilized 1-hydroxybenzotriazole or derivatives thereof for self-contained, single step modifications of amino groups on proteins, peptides, and amines, or to using 1-hydroxybenzotriazole-6-carboxylic acid to effectively and efficiently detect the presence of amines in a sample.